This study was conducted to see whether herpetofaunal assemblage differed amongst hiking trails, undisturbed forest and urban areas within the Margalla Hills National Park, Islamabad Capital Territory, Pakistan. Circular plot area-constrained searches (45 plots in each habitat, each plot with an area of 25 m2) were used from March 2018 to July 2019. We recorded seven amphibian species, nine lizard species and six snake species. The species richness of amphibians and lizards was the same in the studied strata, while the detection and encounter rate of snakes was lower in the undisturbed forest and urban areas. The encounter rate of amphibians differed significantly between urban areas and hiking trails/undisturbed forest. The encounter rate and population density of lizards differed significantly between undisturbed forest and urban areas. The most frequently encountered amphibian species along the hiking trail and urban areas was Duttaphrynus stomaticus, with Hoplobatrachus tigerinus in undisturbed forest. The most common and frequently encountered lizard species along the hiking trail and urban areas was Hemidactylus brookii, while the Ophisops jerdonii was the most frequently seen in undisturbed forest. The most common and frequently encountered snake species along the hiking trail and undisturbed forest was the Indotyphlops braminus, while Ptyas mucosa was the most common in urban areas. The subsequent bio-assessment, based on herpetofauna, of the Park revealed good to excellent biotic integrity The Park faces threats including livestock grazing, alien invasive vegetation and human disturbance due to settlements, restaurants and tourism-related activities. While several of these threats have been mitigated since the establishment of the Islamabad Wildlife Management Board, the Park still requires improved management, especially regarding regulating tourism.